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Microfluidic Assays for High Throughput Screening of Protein Quality in
Bioprocess Development Bahram FathollahiMicrofluidics R&D
CPAC Summer Institute 2009
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What is LabChip Technology?
MiniaturizationIntegrationAutomationActive Fluidic Control
─ Electrokinetics─ Pressure and vacuum
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Photolithography & Etching
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Bioanalyzer 2100 LabChip GX
Higher Throughput
Improved Data Quality
Labor Savings vs. Gels
Digital information
Easy-to-use
Experion
LabChip PlatformsSeparation Assays
RNA
Large Install base (over 5,000 Agilent 2100s).Over 500,000 chips shipped/yr
Protein
DNA
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Need for High Throughput Screening of Antibody Product Quality
There is a clear need for high throughput technologies to address multi-factorial DOE studies
Traditional Assay:e.g. Conventional CE-SDS (to determine fragmentation, purity)
~30 minutes per separation + 20 minutes for rinsing and repriming of capillaryAt best - 20 samples per day (24 h, 1 instrument)
LabChip CE-SDS:40 sec per separation (70X faster than CE-SDS)> 400 samples per day on 1 instrument
“Quality by Design” (QbD) requires deeper understanding between process parameters and outcomes; every parameter choice is an opportunity to design in quality
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Rapid Separations2)/( σLN = Resolution figure of merit: “Theoretical Plates”
0
2
/1112
LLwLN
+⎟⎠⎞
⎜⎝⎛=
DEwL
ep
24
2
0µ
=
Theoretical Plates, considering:• Width of initial sample plug• Diffusion
L0 = Characteristic length scale for “diffusionlimited separations”
Two regimes: L << L0: N = 12(L/w)2
L >> L0, N = µepEL/(2D)
Resolution defined by injection width!
The time needed to achieve certain degree of resolution between peaks (for L=L0) scales with the dimensions of the sample plug width:
t0=w2/24D
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Visualization of loading, injection and of loading, injection and separationseparation
Movie showing loading, injection, and separation of 200 ng/µL of fluorescently tagged alpha-lactalbumin and carbonic anhydrase
20X magnification with panning
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ScreeningTiter – Clone selection– Cell culture optimization
Purity Aggregation (covalently linked) Degree of Glycosylation– % non-glycosylated heavy chain
Fragmentation and Ab assembly Disulfied IsomersCharge heterogeneityPurification process development – Recovery– Process impurities– Product related impurities– Process characterization and
optimization
Analytical Inst. RequirementsRobust, simple to useHigh throughputPredictiveAutomation-friendlyRapid time-to-resultAnalyze crude samples– Clone selection– Cell culture optimization
Minimal sample preparationQuality information – sizing, quantification, and purityHigh resolution
High Throughput Screening Assays
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LabChip SystemWorkflow:
Integrates the entire SDS-PAGE process onto a microfluidic chip
Determine size, quantification, and purity
Automatic sampling from 96- or 384-well plate
Throughput and sample requirements:
Fast sample analysis - 41 seconds per sample
Can run crude samples
96-well plate analyzed in < 1.25 h
Can do >400 samples per day per instrument
2 µl of sample
Minimal sample preparation
21 CFR Part 11 support
Microchip CE
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Features• Integrated sample
processing & analysis• Walk away automation • Easy-to-use software• Enables complex DOE
experiment
Staccato Protein Workstation: Workflow Automation
Scalable design: Twister plate loading, liquid handling sample prep, and fully integrated system
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Protein Assay Specifications
Type Specification
Sizing Range Protein 100 Assay: 14 - 100 kDa
Protein 200 Assay: 14 - 200 kDa ( can detect higher Mw proteins by increasing the separation and sample-load time)
Sizing Accuracy* ± 20%
Resolution* ± 10% difference in MW across the sizing range, 50% valleyResolution is comparable to a 10 cm 4-20% SDS-PAGE gel
Linear Dynamic Range* 5 - 2000 ng/µL
Relative Concentration CV* 30% up to 120 kDa relative to the ladder
Sensitivity* 5 ng/µL (10 ng) Carbonic AnhydraseSensitivity is equivalent to Coomassie stain
Analysis Time per Sample Protein 100 Assay: ~34 seconds/sample
Protein 200 Assay: ~41 seconds/sample
Chip Lifetime 4 96-well plates
Analysis Time Per Plate 96-well plate <1.5 hrs
Maximum Salt Concentration 1M NaCl
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Automated Sample Analysis Results
View individual sample results by selecting wells on the plate map
Digital gel image for quick screening of data, with expected bands flagged
Sizing, concentration, and purity results
The expected proteins displayed as an electropherogram
% purity of LC
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Sample and Chip Preparation
SampleBuffer
+SDS
HeatDenature
Crude or purified Sample
Protein 96- or 384-well plate
Add reagents to chip well
Output in 75 min
for 96-well plate
2µl
Product Quality -Titer, % purity,degree of glycosylation, ….
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Protein Chip
Vacuum is applied to Well 1 through the pressure load channel to aspirate the sample from the well plate, through the sipper and onto the chip
Voltage is applied between Wells 3 and 8 to electrokinetically inject a sample plug
Voltage is then applied between Wells 7 and 10 to perform electrophoretic separation and staining
Electrokinetic destaining is achieved by diluting the separation channel contents from Wells 2 and 9
Detection of proteins occurs just after destain intersection
The microfluidic channels (red) are filled with polymer solution containing SDS and dye.The polymer solution in destain channels is free of SDS and dye
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On-Chip Protein Staining Mechanism
The LabChip protein assay detects proteins using a dye that is fluorescent in a micelle environment (does not directly stain the protein)Therefore “destaining” is required to reduce background signal from free micellesDestaining is accomplished by diluting down the concentration of the SDS and dye Apparent quantum yield of dye is higher in a protein micelle complex than in free micelles
Non-fluorescent dye
fluorescent dye
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Visualization of Destaining
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Effect of Increasing Dilution Ratio
Low destain, high background still above CMC. Increase destaindecrease background and increase protein signal
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Protein Assay Characteristics
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Detection LinearityDilutions of a MAb under non-reducing conditions in the range: 8 µg/mL to 2 mg/mL
8 replicates were run at each concentration
Linear over 3 orders of magnitude
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Low Level Impurity Detection
Impurities of as low as 0.5% to 1% are clearly identified
Lysozyme was spiked into the sample at 1% of total protein and was readily identified, with a S:N of 9:1
LC
HC
NGHC
Ly
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Inter-Assay Precision and Quantification
Experimental Conditions3 different operators3 different chips3 different instruments3 different MAb6 sample preps per operator per MAb
PrecisionRSD of the percent purity of the intact MAb, light chain, heavy chain, and non-glycosylated heavy chain.
High Inter-Assay Precision
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Assay Precision and Quantification
Quantification of intact IgGEach MAb diluted to expected concentration of 1000 ng/µl
Quantification from a standard curve of normalized corrected area versus expected concentration of a MAb
Highly reproducible - Can be used for titer determination
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High Throughput Screening Assays
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High Throughput Screening Assay Traditional Methods vs LabChip CE-SDS
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1
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1. Chen X. et al. Microchip assays for screening monoclonal antibody product quality. Electrophoresis 2008, 29, 4993-5002
2. Xiaooyu Chen and Gregeory Flynn, A high throughput dimer screening assay for monoclonal antibodies using chemical cross-linking and microchip electrophoresis, Journal of chromatography B (2009) in press
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CE-SDS vs LabChip: Reduced
Amgen Publication:
“Microchip CE-SDS… provide sufficient resolution and sensitivity for this purpose but on a time scale approximately 70 times faster (41 s versus 50 min per sample) than conventional CE separation”
Chen X. et al. Microchip assays for screening monoclonal antibody protein quality. Electrophoresis, 29 (2008) 4993-5002
CE-SDS
70 times as fast as CE-SDS with comparable resolution
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5 6 7 8 9 10
Flu
ore
sc
en
ce
Time (seconds)
-50
0
50
100
150
200
250
300
350
400
450
500
550
16 18 20 22 24 26 28 30 32
40 secLabChip 90
CE-SDS vs. LabChip: Non-Reducing
Peak Number
Migration Time,
seconds
Corrected Relative
Area
Migration Time,
minutes
Corrected Relative
Area Delta1 18.6 0.4% 15.5 0.7% 0.3%2 20.5 0.1% 18.7 0.2% 0.1%3 23.9 0.3% 22.2 0.3% 0.0%4 26.4 0.5% 25.2 0.5% 0.0%5 27.8 2.0% 26.7 2.4% 0.4%6 29.0 96.7% 27.8 95.9% 0.8%
Total Impurities 3.3% 4.1% 0.8%
LC-90 CE-SDS
1 2 3 45
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Data is quite consistent, despite differences in:
InstrumentSample preparation bufferDetection methodSome impurities are known to be sensitive to sample preparation conditions
35 minute separationBaselinevariability
Siemiatkoski et al. Use of LabChip 90 in Product Development of a Recombinant Fusion Protein. Protein Summit 2008, London UK
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NFGF NFGF
Crude Harvest Antibodies
Bioreactor Microbioreactor
Heavy Chain [mg/L] 2435 2498
HC purity [%] 52 51
NFGF [%] 3.95 3.99
Bioreactor Microbioreactor
• Heavy chain content and quality is virtually identical between the two platforms.
Bioreactor Microbioreactor
R. Legmann et. al., A predictive high-throughput scale-down model of monoclonal antibody production in CHO cells, Biotechnology & Biengineering DOI 10.1002/bit.22474 (2009) in press
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Analysis of Aggregates
Good resolution and detection of covalently linked aggregatesChen X. et al. Microchip assays for screening monoclonal antibody product quality. Electrophoresis 2008, 29, 4993-5002
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Aggregate Screening AssayCrosslinking chemistry of dimers
Disuccinimidyl suberate NHS esters react with primary amines
1. Flynn G. et. Al. Microchip Assay for Screening Antibody Quality. LabAutomation09, 2009 Palm Springs CA2. Xiaooyu Chen and Gregeory Flynn, A high throughput dimer screening assay for monoclonal antibodies
using chemical cross-linking and microchip electrophoresis, Journal of chromatography B (2009) in press
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Correlation With SECMAbs Crosslinked at pH 10.5
Xiaooyu Chen and Gregeory Flynn, A high throughput dimer screening assay for monoclonal antibodies using chemical cross-linking and microchip electrophoresis Journal of chromatography B (2009) in press
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First Level TitleSecond Level Title
Xiaooyu Chen and Gregeory Flynn, A high throughput dimer screening assay for monoclonal antibodies using chemical cross-linking and microchip electrophoresis Journal of chromatography B (2009) in press
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Dimer Assay on LabChip Technology
Xiaooyu Chen and Gregeory Flynn, A high throughput dimer screening assay for monoclonal antibodies using chemical cross-linking and microchip electrophoresis Journal of chromatography B (2009) in press
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Resolution Enhancement
3. Sample Prep (0.32% LDS)Different separation channel lengths
2. Alternative Sample Prep1.4 cm separation channel
1. Standard Sample Prep1.4 cm separation channel
Fluore
scence
Time (seconds)
-25
0
25
50
75
100
125
150
17.5 18 18.5 19 19.5 20 20.5 21 21.5
0.11% LDS
LC90LCGX LCGX
Blue - 0.11% LDSRed – 0.32% LDS
Blue – 1.4 cmRed – 2.0 cm
Takahashi, M.; Xu, H.; Fathollahi, B.; Holovics, H.; Lacher, N. A. “Optimization of high throughput microchip-CE-SDS for screening monoclonal antibody quality, CEPharm, San Francisco, 2008.
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Sampling of LabChip Users
LabChip System
Quantitative Screening Assays on One Platform
Titer– Total and Intact IgG– Low RSD (~ 2%)
Purity– Reducing and Non-reducing
Degree of Glycosylation– Can resolve NGHC from HC
Fragmentation and Ab assemblyCovalent aggregatesPurification process development– Purity and Yield
Pico Protein Assay – June 2009Glycan Profiling – Q4 2009
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Acknowledgements
Collaborators– Greg Flynn - Amgen– Sharmila Babu - Biogen Idec– Joe Semiatkoski – Biogen Idec– Xiaoyu Chen - Novartis– Nate Lacher – Pfizer– Larbi Rhazi
Caliper Life SciencesAssay Development– Irina Kazakova– Raj Singh
Microfluidics R&D– Hui Xu– Melissa Takahashi
Chip ManufacturingSW Engineering
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First Level TitleSecond Level Title
First Level textSecond Level Text– Third Level Text
– Fourth Level Text
